Cells derived from 47 tumors of breast, ovarian, esophageal, and colorectal origin and 16 paired esophageal biopsies were exposed to anticancer agents (cisplatin; 5-fluorouracil; epirubicin; doxorubicin; paclitaxel; irinotecan and topotecan) in short-term cell culture (6 days). Real-time quantitative PCR was used to measure up- or down-regulation of 16 different resistance/target genes, and when tissue was available, immunohistochemistry was used to assess the protein levels.

In 8/16 paired esophageal biopsies, there was an increase in the expression of multi-drug resistance gene 1 (MDR1) following epirubicin + cisplatin + 5-fluorouracil (ECF) chemotherapy and this was accompanied by increased expression of the MDR-1 encoded protein, P-gp. Following exposure to doxorubicin in vitro, 13/14 breast carcinomas and 9/12 ovarian carcinomas showed >2-fold down-regulation of topoisomerase IIα (TOPOIIα). Exposure to topotecan in vitro, resulted in >4-fold down-regulation of TOPOIIα in 6/7 colorectal tumors and 8/10 ovarian tumors.

Thirty-four esophageal adenocarcinoma biopsies, thirty-two of which were paired samples were obtained from patients (17M:1F; median age 57, range 42–81) before and after administration of 2 cycles of ECF chemotherapy. After enzymatic digestion, tumor derived cells were centrifuged over Ficoll (Sigma Chemical Co, Poole, UK, Cat. No. 1077-1) to remove blood contaminating cells, washed in PBS, and stored in RNA later (Ambion, Huntingdon, UK) at -80°C until further molecular analysis was performed. Tissue sections from these samples were stained for GST-π, MRP1, P-gp and TS. All tumor samples were removed as part of patient treatment, with consent for tissue donation and local research ethics committee approval for use of the tissue surplus to diagnostic requirements for cellular and molecular assays. Chemosensitivity data were available for 9 patients with recurrent ovarian cancer, before treatment and on relapse, though in only one case was material available from both samples for quantitative RT-PCR.

Cisplatin, 5-fluorouracil (5-FU), epirubicin, doxorubicin, irinotecan, paclitaxel (Taxol^®) and topotecan (Hycamptin^®) were obtained from the pharmacy at Queen Alexandra Hospital (Portsmouth, UK). Cisplatin, 5-FU, irinotecan and paclitaxel were stored at room temperature, while all other drugs were stored at -20°C, as previously reported [11]. Test drug concentrations (TDC) were 10.0 μM for cisplatin, 345 μM for 5-FU, 148 μM for irinotecan, 15.9 μM for paclitaxel, 2.5 μM for doxorubicin, 0.862 μM for epirubicin, and 1.64 μM for topotecan. Combinations were made up by adding two or three drugs concurrently at their 200% TDC at the beginning of the ATP-cell viability assay and diluted in a constant ratio: sequential studies were not performed.

Briefly, tumor tissue or fluid was taken by a histopathologist or surgeon under sterile conditions, and transported to the laboratory in cell culture medium of Dulbecco's modified Eagle's medium (DMEM; Sigma Cat No. D5671) with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Sigma, Cat No. P0781) at 4°C. Cells were obtained from solid tumors by enzymatic dissociation, usually 0.75 mg/ml collagenase (Sigma Cat No. C-8051) overnight. Viable tumor-derived cells were purified by density centrifugation (Histopaque 1077-1, Sigma), washed, counted and resuspended to 100,000 cells/ml in case of effusions or 200,000 cells/ml for solid biopsies. In the meantime 96-well polypropylene microplates (Corning-Costar, High Wycombe, UK; Cat No. 3790) were prepared with each drug/combination at six doubling dilutions in triplicate from 200% TDC to 6.25% TDC, according to Andreotti et al. [9]. Approximately 10,000–20,000 cells/well were added to the plates to a final volume of 200 μl/well. The plates were then incubated at 37°C in 5% CO₂ for 6 days, after which the degree of cell inhibition was assessed by measurement of the remaining ATP in comparison with negative control (no drug, MO) and positive control (maximum inhibitor, MI) rows of 12 wells each. Prior to cell lysis with an ATP-extracting reagent, an aliquot of 150 μl of cell suspension were removed from each well, centrifuged, washed with phosphate buffered saline (PBS) and stored at -80°C in a GTIC-containing solution (lysis buffer RA1, Macherey-Nagel, Düren, Germany; Cat. No.740961) until further molecular analysis was carried out. The RNA was subsequently extracted from aliquots that had been exposed to a drug concentration capable of inhibiting cell growth by 40–60%. ATP was extracted from the remaining 50 μl cell suspension and measured by light output in a microplate luminometer (Berthold Diagnostic Systems GmbH, Pforzheim, Germany) following addition of luciferin-luciferase.

Cells obtained after enzymatic dissociation from endoscopic esophageal biopsies or short term cell culture were either resuspended in RNA later (Ambion, Huntingdon, UK; Cat No. 7020) or lysed with buffer RA1 and stored at -80°C until RNA extraction. Total RNA was extracted from at least 50,000 cells with a commercially available kit (NucleoSpin^® RNA II mini, Macherey-Nagel; Cat No. 740955) according to the manufacturer's instructions. The protocol included a DNase digestion step to prevent carry-over of genomic DNA in further analysis.

A two-step protocol was employed. Firstly, total RNA was reverse-transcribed using the Promega reverse transcription system (Promega, Southampton, UK; Cat No. A3500) including 8 μl RNA, 0.5 μg random primers, 20 units of recombinant RNasin^® ribonuclease inhibitor and 15 units of reverse transcriptase AMV (Promega, Cat No. M9004) to each 20 μl reaction. The resulting c-DNA was amplified by qPCR on a Biorad iCycler instrument (BioRad Laboratories, Hemel Hampstead, UK). The constituents of each PCR reaction (25 μl) were 1 μl of cDNA (or H₂O), 200–500 nM of each primer (Table 1), 200 μM each dATP, dCTP, dGTP, 400 μM dUTP, 3.0–5.0 mM MgCl₂, 0.125 units AMPErase^® UNG, 0.625 units of AmpliTaq Gold DNA polymerase and 1 × SYBR Green PCR buffer (all reagents were from Applied Biosystems, Warrington, UK). Product amplification was performed up to 45 PCR cycles, after uracil removal (2 min at 50°C) and polymerase activation (10 min at 95°C). Each two-step PCR cycle comprised denaturing (15 s at 95°C), annealing, and extending (1 min at 60°C). At the end of each run a final melt curve cycle (cooling to 50°C and then increasing stepwise 1°C to 95°C) was performed to exclude the presence of primer-dimer artefacts. At least 3 housekeeping genes were used for each experiment chosen from the following: glyceraldehyde-3 phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), human porphobilinogen deaminase (PBGD), succinate dehydrogenase complex-subunit A (SDHA) and TATA box binding protein (TBP). The internal reference genes were selected due to their relative low abundance in normal tissue [12]. The housekeeping genes were amplified parallel to the target genes in separate wells. When possible, primer sequences (Table 1) were chosen to span exon boundaries to the following targets: breast cancer resistance protein (BCRP), dihydropyrimidine dehydrogenase (DPD), epidermal growth factor receptor (EGFR), excision repair cross-complementing 1 (ERCC1) gene, glutathione-S-transferase isoform π (GST-π), multi-drug resistance gene 1 (MDR1), MLH1, multi-drug resistance related protein 1 (MRP1), multi-drug resistance related protein 2 (MRP2), metallothionein (MT), major vault protein (MVP), thymidine phosphorylase (TP), thymidylate synthase (TS), topoisomerase I (TOPO I), topoisomerase IIα and β (TOPO IIα and TOPO IIβ). Each amplicon was amplified in a separate reaction to prevent competition among multiple sets of primers. A positive control (pooled c-DNA from a variety of human tumors, including breast, ovarian, colorectal and esophageal carcinoma) and negative controls with no template and RT-negative as template were added in every experiment. All assays were run in triplicate. Validation experiments were run to show that the efficiencies of the target and reference genes amplifications were approximately equal, and in the range 95–105%. The PCR cycle number that generated the first fluorescence signal above a threshold (threshold cycle, Ct; 10 standard deviations above the mean fluorescence generated during the baseline cycles) was determined, and a comparative Ct method was then used to measure relative gene expression [13]. The following formula was used to calculate the relative amount of the transcript in the sample: 2^-ΔΔCt, where ΔCt is the difference in Ct between the gene of interest and the mean of the at least two reference genes, and ΔΔCt = ΔCt of drug non-exposed cells – ΔCt of drug-exposed cells, for the ex-vivo experiments, or ΔΔCt = ΔCt of pre-chemotherapy sample – ΔCt of post-chemotherapy sample, for matched tumor biopsies.

The monoclonal antibodies (Abs) for GST-π, MRP1, Pg-p, and TP (Table 2) were detected using the Chemicon IHC Select™ – Immuno Peroxidase secondary detection system (Chemicon International, Chandlers Ford, Southampton, UK, Cat# Det-HP1000) and stained with 3,3' diaminobenzidine (DAB; HD Supplies, Aylesbury, UK, Cat. 4170) on a Dako Autostainer instrument (Dako, Ely, UK). Positive and negative controls were included with each run. Volumes of 50 μl avidin/ml goat serum and 50 μl biotin/ml primary Ab were used to block endogenous avidin binding. The slides were counterstained with Gills Haematoxylin, dehydrated and cleared using the Leica^© XL slide staining machine and mounted in Styrolite^® mounting medium (BDH, Poole, Dorset, UK; Cat No. 361704Y).

IHC was performed on 16 paired esophageal biopsies: qRT-PCR data were obtained for four of these pairs. Overall, we were able to confirm an increased expression of the MDR-1 encoded protein, P-gp (Table 4), in 8/16 paired tumor samples obtained from patients who had been exposed to ECF chemotherapy. TS positivity (Table 4) was found in 10/16 samples obtained before treatment and 10/16 post-chemotherapy specimens; in this instance the modest induction of TS seen at the mRNA level did not reflect an increase in protein level (data not shown). We found GST-π (Table 4) positivity in 5/16 pre-chemotherapy biopsies. In the post-chemotherapy specimens we detected strong expression of GST-π in all but one of the samples. In one case that had been negative at diagnosis we measured a marginal increase of mRNA levels with qRT-PCR. A total of 13/16 pre- and 12/16 post-chemotherapy biopsies were found to be positive for MRP1 (Table 4). Overall, these data show little correlation between the mRNA and the degree of expression of protein levels determined by IHC. Confounding factors may be the presence of non-cancerous cells as mentioned above, but also the nature of IHC, which is at best a semi-quantitative technique.

Of the 47 solid tumor samples studied in short-term cultures, 7 were esophago-gastric, 17 were breast carcinomas, 13 were ovarian carcinomas and 10 were colorectal tumors. A total of 93 experiments were performed, as in most cases the same sample was treated with 2 or more drugs.

Preliminary data obtained from 2 esophageal biopsies indicated an increase of TS levels following 5-FU treatment. Subsequent in vitro experiments performed on material derived from 10 colorectal tumor samples and 13 breast tumor samples confirmed the expected increase of TS levels (Table 7) in the samples exposed to 5-FU (p < 0.0001, Wilcoxon; Fig. Fig.3f).3f). The results indicated a general trend towards DPD down-regulation in the cells that had been exposed to 5-FU in vitro, though this effect was modest in most cases and we observed a greater than 2-fold decrease in only 7/13 breast samples and 2/10 colorectal samples.

We were able to study tumor-derived cells from 7 esophageal cancer patients, 4 of which had already been given ECF in vivo. The results mirror those obtained from paired esophageal biopsies: we noticed increased expression of TS in the cells that had been exposed to ECF (Fig (Fig3h).3h). However, the expected up-regulation of MDR1 was only detected in 4/7 samples (data not shown), and may therefore be occurring in non-neoplastic cells that do not normally express MDR1, rather than in the neoplastic cells, which we found to express this molecule to the same degree pre- and post-treatment.

There are few studies comparing gene expression before and after chemotherapy. A few studies have reported microarray data in biopsy material taken before and after (or even during) chemotherapy. These studies are of great interest, though normal cell effects cannot be excluded from the results. Buchholz et al. [35] employed cDNA microarray to measure gene expression changes during chemotherapy in 5 patients with breast cancer. Clarke et al. [36] studied gene expression changes in 18 rectal cancer patients undergoing therapy with Mitomycin C or 5-FU. This study reported a number of genes implicated in protein synthesis and RNA metabolism to be significantly decreased during drug treatment. These studies are not directly comparable: tumors of different types respond better to different drugs, and differences in their adaptation to these drugs are therefore expected on the basis of their innate sensitivity or resistance.